Separation of enzymes from microorganism crude extracts by free‐flow zone electrophoresis

Continuous, single‐step, state‐of‐the‐art preparative separations of enzymes from microorganism crude extracts by free‐flow zone electrophoresis are presented. In the first example, the enzymes formate dehydrogenase, formaldehyde dehydrogenase, and methanol oxidase were continuously separated from Candida boidinii crude extract. Yields of 85% to 95% and purification factors between 3 and 7 were obtained along with a simultaneous separation of the finer cell debris from the enzymes. Using multiple injections of sample, a throughput of 46.2 mg protein/h was recorded. In the second example, a fivefold purification of β‐galactosidase from Escherichia coli was achieved along with complete, simultaneous cell debris separation from the enzyme. The yield of the enzyme was greater than 90%. The preparative free‐flow zone electrophoresis experiments were run continuously for a period of 12 h and the separations were found to be stable; i.e., the enzymes and the cell debris eluted at their respective fraction numbers during the entire period. In both examples, choice of the type of buffer played a critical role and had to be investigated and optimized experimentally. Scale‐up aspects of the separations are also discussed. Recently, by comparison of free‐flow zone electrophoresis with ion‐exchange chromatography, we have presented evidence that free‐flow electrophoresis separations are governed by net surface charge (S. Nath et al., Biotechnol. Bioeng. 1993, 42 : 829–835). Here, we offer further confirmation of this evidence by comparison of preparative free‐flow zone electrophoresis experiments at various pHs on a mixture of two model proteins with analytical electrophoretic titration curves of the proteins. We are thus in a position to predict separations in free‐flow zone electrophoresis. © 1996 John Wiley & Sons, Inc.